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TitleStructure and evolution-guided design of minimal RNA-guided nucleases.
Journal, issue, pagesScience, Vol. 393, Issue 6808, Page 313-318, Year 2026
Publish dateJul 16, 2026
AuthorsPetr Skopintsev / Isabel Esain-Garcia / Evan C DeTurk / Peter H Yoon / Zehan Zhou / Trevor Weiss / Maris Kamalu / Ajit Chamraj / Kenneth J Loi / Conner J Langeberg / Ron S Boger / Hunter Nisonoff / Hannah M Karp / Lin-Xing Chen / Honglue Shi / Kamakshi Vohra / Jillian F Banfield / Jamie H D Cate / Steven E Jacobsen / Jennifer A Doudna /
PubMed AbstractThe design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic ...The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.
External linksScience / PubMed:42462008
MethodsEM (single particle)
Resolution2.8 Å
Structure data

73644

9yyg

EMDB-73644, PDB-9yyg:
AI-generated RNA-guided nuclease TAM-bound state
Method: EM (single particle) / Resolution: 2.8 Å

73645

9yyh

EMDB-73645, PDB-9yyh:
AI-generated RNA-guided nuclease R-loop formed state
Method: EM (single particle) / Resolution: 2.8 Å

Source
  • synthetic construct (others)
  • deinococcus radiodurans (radioresistant)
KeywordsRNA BINDING PROTEIN / CRISPR / nuclease / AI / Evolution Scale Modeling / Inverse Folding / ESM-IF1 / TnpB / TAM / PAM / spacer / exonuclease / DNA binding protein-DNA complex / enzyme / ribonucleoprotein / RNA / RNA-guided nuclease / Cas12 / ISDra2 / ESM

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